In the central nervous system, astrocytes form tripartite synapses with presynaptic and postsynaptic neuronal terminals and can participate in bidirectional communication with neurons through secretion of 'gliotransmitters' such as glutamate, ATP, and D-serine that can impact neuronal activity. Previous studies have suggested that a major mechanism mediating these secretion events is vesicular exocytotic release evoked by elevations in intracellular calcium (Ca2+) levels. However, the molecules and pathways involved in generating these Ca2+- dependent events remain poorly understood. Although astrocytes are electrically non-excitable cells, they exhibit a form of excitability based on elevations of cytoplasmic Ca2+ levels in response to physiological and pathological signals. This is primarily mediated by the release of Ca2+ from the endoplasmic reticulum (ER) after activation of ER inositol 1,4,5 triphosphate (IP3) receptors downstream of numerous G-protein-coupled cell surface receptors (GPCRs). Importantly, the emptying of ER Ca2+ stores activates a secondary Ca2+ influx mechanism known as store-operated Ca2+ entry (SOCE) through store-operated channels (SOCs). This proposal seeks to examine the functional relevance of SOCE to astrocyte gliotransmitter exocytosis in order to gain mechanistic insight into how astrocytes can modulate synaptic physiology. Preliminary evidence indicates that the SOCs that give rise to a major route of Ca2+ entry in astrocytes are the Ca2+ release-activated Ca2+ (CRAC) channels arising from the canonical Orai1-STIM1 proteins. Further, preliminary experiments show that CRAC channels mediate purinergic GPCR-activated Ca2+ signals in astrocytes and that activation of SOCE stimulates exocytosis and ATP secretion from astrocytes. Therefore, the central hypothesis of this project is that CRAC channels regulate gliotransmission and enable astrocyte-neuron communication by serving as a vital Ca2+ delivery pathway in astrocytes. This hypothesis will be addressed through two specific aims that will employ a multidisciplinary approach combining imaging, electrophysiological, and biochemical approaches as well as genetically engineered mice lacking CRAC channel function. Aim 1 will define the functional role of CRAC channels for gliotransmitter exocytosis. Aim 2 will seek to determine the significance of CRAC channels for astrocyte modulation of synaptic transmission. Findings from these aims will reveal the role of a Ca2+ signaling pathway important for regulating Ca2+ homeostasis and effector functions in astrocytes. Since astrocytes have specialized access to synapses, this signaling pathway may be a valuable target for the development of novel therapeutics for pathological diseases affecting synaptic function.